Sleep is an essential, evolutionarily conserved process which, if unfulfilled, contributes to human pathology. The importance of sleep is underscored by its tight homeostatic control: sleep drive increases with time spent awake and dissipates with time spent asleep. However, the molecular mechanisms underlying regulation of sleep and the neural circuitry that controls sleep homeostasis are largely unknown. The fruit fly, Drosophila melanogaster, which has proven useful for identifying genes involved in behavior, human health and disease, has also emerged as a valuable genetic model system for studying sleep. Using a forward genetic screen, we identified the novel gene sleepless (sss) that is required for both baseline and homeostatic recovery sleep following sleep deprivation. In sss mutants, we found that levels of the sleep-regulating K channel, Shaker (Sh), are reduced, leading to the hypothesis that sss couples sleep drive to lowered membrane excitability. More recently we have also shown that sss can regulate the localization of Sh channels in addition to both amplitude and kinetics of Sh currents. Consistent with direct regulation of Sh by SSS, we have demonstrated that Sh expression is promoted post-transcriptionally via the formation of a stable complex between channel and SSS. sss is under the control of RNA editing machinery, and edited sss is less effective than uneditable sss at promoting sleep. We thus hypothesize that RNA editing controls the ability of SSS to interact with Sh, thereby altering activity and subcellular trafficking of the channel. The structural basis for SSS-Sh interactions is particularly intriguing: SSS is one of the founding members of a large family of relatively uncharacterized proteins that resemble neurotoxins, which often act on ion channels, raising the possibility that other members of this family may regulate excitability and sleep. The focus of this proposal is to determine the molecular basis of sleep regulation by sss, particularly with regard to Sh, and to describe the neural circuitry involved. The specific aims are to: 1) determine mechanisms by which sss regulates Sh, 2) determine the role of RNA-editing of sss in modulation of sleep and Sh currents, and 3) determine where in the brain sss acts to regulate sleep. Collectively these studies will improve our understanding of the molecular basis of sleep need and how it leads to major changes in electrical activity in the brain. Such findings may also help identify new targets for intervening both in disorders of sleep and in disorders related to misregulation of neuronal excitability in general. PUBLIC HEALTH RELEVANCE: The proposed studies will improve our understanding of the molecular basis of sleep need and how it leads to major changes in electrical activity in the brain. Such findings will help identify new targets for intervening both in disorders of sleep and in disorders related to misregulation of neuronal excitability in general.